Gas-Phase Hydrogen/Deuterium Scrambling in Negative-Ion Mode Tandem Mass Spectrometry

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a powerful method to characterize protein conformational dynamics. Workflows typically utilize pepsin digestion prior to MS analysis to yield peptide level structural resolution. Tandem mass spectrometry (MS/MS) can potentially facilitate determination of site-specific deuteration to single-residue resolution. However, to be effective, MS/MS activation must minimize the occurrence of gas-phase intramolecular randomization of solution-generated deuterium labels. While significant work has focused on understanding this process in positive-ion mode, little is known about hydrogen/deuterium (H/D) scrambling processes in negative-ion mode. Here, we utilize selectively deuterated model peptides to investigate the extent of intramolecular H/D scrambling upon several negative-ion mode MS/MS techniques, including negative-ion collision-induced dissociation (nCID), electron detachment dissociation (EDD), negative-ion free radical–initiated peptide sequencing (nFRIPS), and negative-ion electron capture dissociation (niECD). H/D scrambling was extensive in deprotonated peptides upon nCID and nFRIPS. In fact, the energetics required to induce dissociation in nCID are sufficient to allow histidine C-2 and C β hydrogen atoms to participate in the scrambling process. EDD and niECD demonstrated moderate H/D scrambling with niECD being superior in terms of minimizing hydrogen migration, achieving ~ 30% scrambling levels for small c -type fragment ions. We believe the observed scrambling is likely due to activation during ionization and ion transport rather than during the niECD event itself.